The purpose of this proposal is to understand the mechanism of replication of picornaviruses typified by poliovirus with special emphasis on the involvement of host protein in viral RNA replication. The roles of viral polymerase and host factor (HF) in the poliovirus RNA-directed synthesis of minus RNA will be studied using both biochemical and serological techniques. Attempts will be made to prepare monoclonal antibodies to different viral- and host-proteins relevant to viral RNA synthesis. Initiation of viral RNA synthesis will be studied by comparing the oligo (U) primed RNA synthesis to the HF catalyzed copying of viral RNA. Effect of anti HF antibody on these systems will be examined. Mechanism of initiation will further be examined of 5'-end analysis of newly synthesized RNAs. Specific questions regarding the mechanism of viral RNA synthesis will be asked using anti HF- and anti polymerase antisera on the in vitro RNA synthesizing system as well as in crude cell extracts. Monospecific antibodies will be used as probes to study the organication of the poliovirus replicase machinery. In vitro reconstitution between HF and viral polymerase (and other viral proteins) will help to understand the structure-function relationship of different proteins present in replicase complex. Viral template RNA recognition by the replicase will be examined using a nitrocellulose filter binding assay. The replicase binding site will be isolated and nucleotide sequence will be determined using available RNA sequencing methods. Attempts will be made to achieve specific transcription of Polio RNA by adding different fractions from infected and/or uninfected cells to the reconstituted system. Finally purified host-factor and anti HF antiserum will be used to determine the role of HF in normal HeLa cells. The long-term goal of this research is to understand the mechanism of picornavirus replication. Studies will be continued to understand the mechanism of viral RNA (Plus RNA) synthesis on minus RNA template. The knowledge of how poliovirus replicates will certainly help to understand the mode of replication of other picornaviruses, especially that of rhinoviruses (causative agent of common cold).